Role of intracellular calcium in neuronal death following cerebral ischemica: Neuroprotection by dantrolene and BCL-2

Increased cytosolic concentrations of {dollar}rm Casp{lcub}2+{rcub} (lbrack Casp{lcub}2+{rcub}rbracksb{lcub}I{rcub}){dollar} have been implicated in neuronal death from cerebral ischemia. This dissertation research investigated how cerebral ischemia alters various VDCC and ADCC and the possible contribution of these change to selective and delayed ischemic neuronal death.; Changes of various VDCC (L- and N-type) or ADCC (NMDA, AMPA, kainate and metabotropic glutamate receptors) in canine brains following global cerebral ischemia induced by 10 min cardiac arrest and during 0.5, 2, 4, or 24 hr reperfusion were studied by measuring binding of radiolabeled agonists or antagonists to these receptors. The binding of {dollar}rmlbracksp{lcub}125{rcub}Irbrackomega{dollar}-conotoxin GVIA to N-type VDCC in different brain regions was not significantly different from sham controls at any time point following cardiac arrest. {dollar}rmlbracksp3Hrbrack{dollar} PN200-110 to L-type VDCC binding was decreased in each region immediately following ischemia, recovering within 0.5 hr of recirculation. These results suggest that increased binding or number of L- and N-type VDCC may not be a main cause of neuronal death during early hours of reperfusion after cerebral ischemia. Elevated binding or number of NMDA but not non-NMDA glutamate receptors (AMPA, kainate and metabotropic glutamate receptors) during early reperfusion period may be one of the reasons for increased {dollar}rmlbrack Casp{lcub}2+{rcub}rbracksb{lcub}I{rcub},{dollar} and neuronal death in striatum, and early use of NMDA receptor antagonists after cerebral ischemia would be expected to have neuroprotective effects in this region.; To investigate the role of {dollar}rm Casp{lcub}2+{rcub}{dollar} release from ER in neural cell apoptosis, an in vitro cell death model was established in GT1-7 hypothalamic neurosecretory cells by treating them with thapsigargin. This drug selectively inhibits ER {dollar}rm Casp{lcub}2+{rcub}{dollar} ATPase, inhibiting {dollar}rm Casp{lcub}2+{rcub}{dollar} transport from the cytosol into the ER and causing a net {dollar}rm Casp{lcub}2+{rcub}{dollar} release from ER. Thapsigargin-induced GT1-7 cell death was characterized by nuclear condensation and DNA fragmentation, and was attenuated by the protein synthesis inhibitor cycloheximide. These characteristics are features of apoptosis. These results suggest abnormal {dollar}rm Casp{lcub}2+{rcub}{dollar} release from ER may induce apoptosis in neurons.; Dantrolene, which inhibits {dollar}rm Casp{lcub}2+{rcub}{dollar} release from ER by blocking release from the ryanodine receptor channel, significantly inhibited thapsigargin-induced elevation of {dollar}rm Casp{lcub}2+{rcub},{dollar} DNA fragmentation and cell death in GT1-7 cells in a dose-dependent manner. These results suggest that abnormal {dollar}rm Casp{lcub}2+{rcub}{dollar} release from ER plays an important role in delayed neuronal apoptosis after cerebral ischemia and drugs such as dantrolene which block {dollar}rm Casp{lcub}2+{rcub}{dollar} release from ER may be good candidates for neuroprotection after cerebral ischemia.; Overexpression of bcl-2 in GT1-7 cells also significantly inhibited nuclear condensation, DNA fragmentation and cell death induced by thapsigargin or caffeine. However, bcl-2 could not suppress the elevation of {dollar}rmlbrack Casp{lcub}2+{rcub}rbracksb{lcub}I{rcub},{dollar} induced by thapsigargin, suggesting that it may inhibit GT1-7 cell death at a point downstream from ER Ca{dollar}sp{lcub}2+{rcub}{dollar} release. These results further support that bcl-2 may inhibit a common biochemical pathway for induction of apoptosis.